PXD023419-3

PXD023419 is an original dataset announced via ProteomeXchange.

Title	MS-based sequencing of the anti-FLAG-M2 antibody
Description	We demonstrate a method for direct de novo sequencing of monoclonal IgG from the purified antibody products. The method uses a panel of multiple complementary proteases to generate suitable peptides for de novo sequencing by LC-MS/MS in a bottom-up fashion. Furthermore, we apply a dual fragmentation scheme, using both stepped high-energy collision dissociation (stepped HCD) and electron transfer high-energy collision dissociation (EThcD) on all peptide precursors. The method achieves full sequence coverage of the monoclonal antibody Herceptin, with an accuracy of 98% in the variable regions. We applied the method to sequence the widely used anti-FLAG-M2 mouse monoclonal antibody, which we successfully validated by remodeling a high-resolution crystal structure of the Fab and demonstrating binding to a FLAG-tagged target protein in Western blot analysis. The method thus offers robust and reliable sequences of monoclonal antibodies.
HostingRepository	PRIDE
AnnounceDate	2021-06-16
AnnouncementXML	Submission_2021-06-15_22:42:01.461.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Joost Snijder
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090; scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	2-pyrrolidone-5-carboxylic acid (Glu); 2-pyrrolidone-5-carboxylic acid (Gln); monohydroxylated residue; iodoacetic acid derivatized residue; complex glycosylation; deamidated residue
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2021-01-05 05:33:34	ID requested
1	2021-01-10 23:32:05	announced
2	2021-06-15 22:40:48	announced	2021-06-16: Updated project metadata.
⏵ 3	2021-06-15 22:42:02	announced	2021-06-16: Updated project metadata.

Peng W, Pronker MF, Snijder J, Sequencing of Monoclonal Antibodies Using Multiple Proteases and a Dual Fragmentation Scheme. J Proteome Res, 20(7):3559-3566(2021) [pubmed]

submitter keyword: antibody, stepped HCD, EThcD, de novo sequencing

Joost Snijder
contact affiliation	Utrecht University
contact email	j.snijder@uu.nl
lab head
Joost Snijder
contact affiliation	Utrecht University
contact email	joost.snijder@gmail.com
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2021/01/PXD023419

PRIDE project URI

• PRIDE
  1. PXD023419
     1. Label: PRIDE project
     2. Name: MS-based sequencing of the anti-FLAG-M2 antibody